TITLE kHz-rate in vivo imaging of neural activity throughout the living brain SUMMARY The overarching challenge in neuroscience today is how to monitor the neural signaling events in intact brains of behaving animals at synaptic or cellular spatial resolution and millisecond time resolution. Multiphoton fluorescence microscopy stands out among the existing brain imaging methods because of its ability to visualize the neuronal signals in optically opaque brains at sub-micron spatial resolution. However, hampered by the fundamental speed limitation of the gold-standard mechanical scanning mirrors, current multiphoton fluorescence microscopy approaches run short of imaging speed required to record the neuronal dynamics at millisecond resolution in an extended volume of brain. The Tsia lab has recently developed an ultrafast laser-scanning imaging technique called free-space angular-chirp-enhanced delay (FACED). Bypassing the use of the mechanical scanning mirrors and their speed limitation, a FACED module can achieve ultrafast line-scanning rate beyond multi-MHz. Employing FACED for imaging modalities such as coherent microscopy and one-photon fluorescence microscopy, the Tsia lab has demonstrated single-cell dynamics monitoring at a 2D frame rate of 3,000 frames per second. In this proposal, building upon the recent demonstrations of FACED imaging in Tsia lab, the expertise in multiphoton in vivo brain imaging of the Ji lab, and the computational and statistical analysis toolboxs developed by the Paninski lab, we aim to integrate FACED with the established as well as emerging methods of multiphoton fluorescence microscopy to achieve ultrafast in vivo kHz-rate recording of neural signaling events throughout the living brain and validate the proposed approaches in the context of real- world neurobiological enquiries.